Histopathologic changes in macaques with an acquired immunodeficiency syndrome (AIDS).

The authors recently described the clinical course of an Acquired Immunodeficiency Syndrome (AIDS) in a colony of macaque monkeys. In the present study, they have reviewed the histopathology of tissues obtained from a cohort of 16 animals with this clinical syndrome at necropsy. They found evidence in these animals of several opportunistic infections, including cytomegalovirus (CMV), simian virus 40 (SV-40), and noma. Furthermore, a number of other unusual pathologic processes were noted. In 4 animals an array of lymphoproliferative disorders was observed, ranging from multiple nodules of lymphocytes in the kidney, liver, and bone marrow, to frank lymphoma. Evidence of retroperitoneal fibrosis was found in 3 of the animals. Finally, amyloidosis was observed in several animals; in two instances it was present only in the mucosa of the small intestine.     

Viral evolution and challenges in the development of HIV vaccines

Potent virus-specific cytotoxic T lymphocyte (CTL) responses elicited by candidate AIDS vaccines have been shown to provide short-term control of viral replication following pathogenic viral challenges in rhesus monkeys. We have recently shown that vaccines that control rather than prevent immunodeficiency virus infections are still subject to immune escape. In particular, viral mutations can develop that result in viral escape from recognition by immunodominant CTL, loss of immune control of viral replication, and clinical disease progression. These data suggest that viral escape from CTL may prove to be a significant limitation of the current generation of CTL-based AIDS vaccines.     

Vaccine protection against simian immunodeficiency virus infection.

Rhesus monkeys were immunized by multiple inoculations with purified, disrupted, noninfectious simian immunodeficiency virus (SIV) in adjuvant. Immunized monkeys developed anti-SIV antibodies detectable by whole-virus ELISA and by immunoblot reactivity; these antibodies had weak neutralizing activity. One week after the last immunization, monkeys were challenged with 200-1000 animal infectious doses of uncloned, live SIV. The same strain of SIV that was used for vaccination was also used for challenge. Anamnestic antibody responses and SIV recovery from peripheral blood were used to evaluate infection following the live virus challenge; two of six vaccinated monkeys showed no evidence of infection following the live virus challenge. Transfusion of 10 ml of whole blood from these two into uninfected, naive rhesus monkeys did not result infection of the recipients, providing further support for the lack of infection in the two previously vaccinated animals. Four of four unvaccinated control monkeys inoculated with these doses of live SIV became infected and three of these died with AIDS 118-258 days after infection. Only one of the six vaccinated monkeys has died to date. In situ hybridization with lymph node biopsy specimens suggested that the virus load was much higher in control macaques than in vaccinated macaques. These results indicate that vaccination with inactivated whole virus can protect macaques against challenge with live SIV. Furthermore, they provide hope that vaccine protection against human AIDS virus infection may be possible.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

Research on tuberculosis and leprosy was revolutionized by the development of a plasmid transformation system in the fast-growing surrogate, Mycobacterium smegmatis. This transformation system was made possible by the successful isolation of a M. smegmatis mutant strain mc²155, whose efficient plasmid transformation (ept) phenotype supported the replication of Mycobacterium fortuitum pAL5000 plasmids. In this report, we identified the EptC gene, the loss of which confers the ept phenotype. EptC shares significant amino acid sequence homology and domain structure with the MukB protein of Escherichia coli, a structural maintenance of chromosomes (SMC) protein. Surprisingly, M. smegmatis has three paralogs of SMC proteins: EptC and MSMEG_0370 both share homology with Gram-negative bacterial MukB; and MSMEG_2423 shares homology with Gram-positive bacterial SMCs, including the single SMC protein predicted for Mycobacterium tuberculosis and Mycobacterium leprae. Purified EptC was shown to bind ssDNA and stabilize negative supercoils in plasmid DNA. Moreover, an EptC–mCherry fusion protein was constructed and shown to bind to DNA in live mycobacteria, and to prevent segregation of plasmid DNA to daughter cells. To our knowledge, this is the first report of impaired plasmid maintenance caused by a SMC homolog, which has been canonically known to assist the segregation of genetic materials.The genetic bases for signature phenotypes of Mycobacterium tuberculosis, including acid fast staining, virulence, and susceptibility to tuberculosis (TB)-specific drugs were unknown before the generation of a plasmid transformation system in mycobacteria (1). Whereas recombinant DNA technology in Escherichia coli laid the foundation for modern genetic research, analysis of mycobacterial genes in E. coli was insufficient to elucidate phenotypes. The profound difference in cell wall composition, lipid metabolism, promoter recognition, and posttranslational modification between mycobacteria and E. coli greatly limited the use of E. coli as a surrogate host for mycobacterial gene analysis. Even though plasmid transformation in Streptomyces, phylogenetically closer than E. coli to Mycobacterium, was established in 1978 (2), neither Streptomyces nor E. coli is sensitive to TB-specific drugs such as isoniazid, ethambutol, ethionamide, thioacetazone, or isoxyl. Snapper et al. developed the first plasmid transformation system for mycobacteria by isolating a Mycobacterium smegmatis mutant, namely mc²155, that allowed for the replication of Mycobacterium fortuitum plasmids (1, 3). This enabled the development of efficient cloning vectors, the recreation of drug-resistant phenotypes, and important discoveries of the biology of mycobacteria. Although mc²155 was first isolated in 1988 and characterized to be a plasmid-specific phenotype in 1990 (4), the genetic basis for this efficient plasmid transformation (ept) phenotype has not been known. Here we are reporting that the mutation mediating the ept phenotype maps to a gene encoding a structural maintenance of chromosomes (SMC) homolog, which plays an important role in cell division—a fundamental cellular process across all domains of life.Cell division requires DNA replication followed by faithful segregation of genetic material to daughter cells. Eukaryotic cells rely on spindle fibers to move homologous chromosomes to the opposite ends of the cell (5, 6). Segregation in prokaryotes resembles that in eukaryotes regarding the basic mechanism and the requirement for SMC proteins. Since the discovery of SMC proteins, extensive studies have led to the understanding of their roles in chromosome condensation and segregation (7). SMC proteins were first functionally characterized in Saccharomyces cerevisiae when a mutant allele smc1-1 caused a chromosome nondisjunction (2:0 segregation) defect at least 10 times more often than observed in the WT strains (8, 9). SMC proteins are highly conserved in eukaryotes and their homologs are found in almost all Archaea, Gram-positive bacteria, and in about 40% of Gram-negative bacteria. Gram-negative organisms, such as E. coli, do not encode the canonical SMC, but rely on a functional analog MukB (10).In prokaryotes, chromosome segregation has mainly been studied by genetic and biochemical analyses of the low-copy number plasmid DNA (11–13). High-copy number plasmids rely primarily on passive diffusion for plasmid maintenance (14), which is inapplicable to chromosomal DNA. However, the accurate segregation of low-copy number plasmids requires dedicated partitioning loci consisting of three components: a cis-acting centromere site and two trans-acting proteins ParA and ParB (15). The mycobacterial pAL5000 is a low-copy number plasmid (two to five copies per cell) (16) and, noticeably, does not require a dedicated plasmid partitioning system for its segregation in M. smegmatis mc²155. Thus, maintenance of pAL5000 plasmids by mc²155, but not by its parental strain, motivated us to look for an alteration in the host machinery responsible for plasmid segregation.In this study, we report that the genetic basis of M. smegmatis mc²155’s ability to segregate episomal plasmids, and thus assume transformability, is a mutation in a gene annotated here as eptC. Biochemical and genetic analyses of EptC demonstrate that the protein belongs to the family of SMC proteins. EptC interferes with faithful segregation of pAL5000 plasmids to daughter M. smegmatis cells by directly binding to plasmids and modulating plasmid supercoiling status. The appropriate supercoiling status has been shown crucial for successful plasmid segregation (17).     

Evaluation of a prime-boost vaccine schedule with distinct adenovirus vectors against malaria in rhesus monkeys

A vaccine that elicits both specific antibodies and IFN-γ-producing T cells is required to protect against pre-erythrocytic malaria. Among the most promising approaches to induce such complex immunity are heterologous prime-boost vaccination regimens, in particular ones containing live viral vector. We have demonstrated previously that adenovectors serotype 35 (Ads35) encoding the circumsporozoite (CS) antigen or liver-stage antigen-1 (LSA-1) are highly effective in improving the T-cell responses induced by immunizations with protein-based vaccines in a heterologous prime-boost schedule. Here we evaluated the potential of a heterologous prime-boost vaccination that combines the Ad35.CS vector with the serologically distinct adenovector Ad5.CS, in rhesus macaques, after establishing the potency in mice. We show that the heterologous Ad35.CS/Ad5.CS prime-boost regimen elicits both antibody responses and robust IFN-γ-producing CD8⁺ T-cell responses against the CS antigen. Analysis of the quality of the antibody responses in rhesus macaques, using indirect immunofluorescence assay (IFA) with Plasmodium falciparum-coated slides, demonstrated that this heterologous prime-boost regimen elicits a high titer of antibodies that are able to bind to P. falciparum sporozoites. Level of the IFA response was superior to the response measured with sera of an adult human population living in endemic malaria region. In conclusion, the combination of Ad35.CS, a vaccine based on a rare serotype adenovirus, with Ad5.CS or possibly another adenovector of a distinct serotype, induces a complex immune response that is required for protection against malaria, and is thus a highly promising approach for pediatric vaccination.     

Modulation of plasmid DNA vaccine antigen clearance by caspase 12 RNA interference potentiates vaccination

The magnitude of the immune responses elicited by plasmid DNA vaccines might be limited, in part, by the duration of vaccine antigen expression in vivo. To explore strategies for improving plasmid DNA vaccine efficacy, we studied the apoptotic process in myocytes of mice vaccinated intramuscularly. We found that after vaccination, the proapoptotic protein caspase 12 (Casp12) was upregulated in myocytes coincident with the loss of vaccine antigen expression. To harness this observation to improve plasmid DNA vaccine efficacy, we used RNA interference technology, coadministering plasmid DNA expressing a short hairpin RNA (shRNA) of Casp12 with plasmid DNA vaccine constructs. This treatment with shRNA Casp12, administered twice within the first 10 days following vaccine administration, increased antigen expression 7-fold, the antigen-specific CD8(+) T cell immune response 6-fold, and antigen-specific antibody production 5-fold. This study demonstrates the critical role for Casp12 in plasmid DNA vaccine-induced immune responses and shows that increased antigen expression mediated by down-modulation of Casp12 can be used to potentiate vaccine efficacy.